Hydrogen Sulfide Attenuates High Glucose-Induced Human Retinal Pigment Epithelial Cell Inflammation by Inhibiting ROS Formation and NLRP3 Inflammasome Activation

Abstract

Hydrogen sulfide (H₂S) has been shown to protect against oxidative stress injury and inflammation in various high glucose-induced insult models. However, it remains unknown whether H₂S protects human retinal pigment epithelial cells (RPE cells) from high glucose-induced damage. In the current study, cell viability, proinflammatory cytokines, ROS, and inflammasome markers were compared in a low glucose- and high glucose-induced cell culture system. The antioxidant N-acetylcysteine (NAC), NLRP3 siRNA, and NaHS were used to test RPE cell responses. The results demonstrate that compared with the low-glucose culture, high glucose triggered higher cell death and increased IL-18 and IL-1β mRNA expression and protein production. Furthermore, high glucose increased the mRNA expression levels of NLRP3, ACS, and caspase-1. Notably, NAC, a ROS scavenger, could attenuate high glucose-induced ROS formation and IL-18 and IL-1β mRNA and protein expression and block inflammasome activation. Silencing the NLRP3 gene expression also abolished IL-18 and IL-1β mRNA and protein expression. Intrudingly, H₂S could ameliorate high glucose-induced ROS formation, IL-18 and IL-1β expression, and inflammasome activation. Taken together, the findings of the present study have demonstrated that H₂S protects cultured RPE cells from high glucose-induced damage through inhibiting ROS formation and NLRP3 inflammasome activation. It might suggest that H₂S represents a potential therapeutic target for the treatment of diabetic retinopathy.

Figure 1

The high-glucose condition decreased cell viability and induced proinflammatory cytokine IL-18 and IL-1β production in ARPE-19 cells. ARPE-19 cells were treated with high or low glucose for 48 hours. (a) Cell viability determined by the CCK-8 assay in the low-glucose group, high-glucose group, high-glucose+H₂S group, and high-glucose+NAC group. (b) Protein levels of IL-18 detected by ELISA. (c) Protein levels of IL-1β detected by ELISA. (d) The mRNA expression levels of IL-18 determined by RT-PCR. (e) IL-1β mRNA levels measured by RT-PCR. Data were presented as the mean ± standard error (SE) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01, compared between the low-glucose and high-glucose groups (low glucose: 5.5 mM, high glucose: 25 mM).

Figure 2

The high-glucose condition increased intracellular ROS formation in ARPE-19 cells. ARPE-19 cells were treated with high or low glucose for 48 hours. ROS production was measured by FCM. (a) FACS results of low-glucose culture condition. (b) FAC results of high-glucose culture condition. (c) A bar graph of average results obtained from three individual experiments. Data were presented as the mean ± standard error (SE) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01, compared between the low-glucose and high-glucose groups (low glucose: 5.5 mM, high glucose: 25 mM).

Figure 3

The high glucose-induced activation of the inflammasome in ARPE-19 cells. The mRNA expression of NLRP3, ASC, and caspase-1 in ARPE-19 cells after low-glucose and high-glucose culture for 48 hours was determined by RT-PCR. (a) NLRP3 mRNA levels were relative to GAPDH. (b) ASC mRNA levels were relative to GAPDH. (c) Caspase-1 mRNA levels were relative to GAPDH. Data were presented as the mean ± standard error (SE) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01, compared between the low-glucose and high-glucose groups (low glucose: 5.5 mM, high glucose: 25 mM).

Figure 4

N-Acetylcysteine (NAC) attenuated intracellular ROS formation and inflammatory response. The inhibition of ROS by NAC decreased the gene expression of NLRP3, ASC, caspase-1, IL-18, and IL-1β and the protein expression of IL-18 and IL-1β in ARPE-19 cells. ARPE-19 cells were cultured under low or high glucose with or without 2.5 mM of NAC for 48 hours. The mRNA and protein expression levels of proinflammatory cytokines IL-18 and IL-1β and inflammasome activation markers NLRP3, ACS, and caspase-1 were determined, accordingly. (a) IL-18 mRNA; (b) IL-1β mRNA; (c) intracellular IL-18 protein levels; (d) intracellular IL-1β protein levels; (e) NLRP3 mRNA; (f) ASC mRNA; (g) caspase-1 mRNA. Data were presented as the mean ± standard error (SE) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01, compared between the high-glucose and high-glucose+NAC groups (low glucose: 5.5 mM, high glucose: 25 mM).

Figure 5

The knockdown of NLRP3 gene expression ameliorated high glucose-induced proinflammatory cytokine production and inflammasome activation. The gene expression levels of IL-18 and IL-1β and inflammasome markers NLRP3, ASC, and caspase 1 were measured in ARPE-19 cells with either NLRP3-siRNA or scrabbling siRNA transfection in low- and high-glucose culture for 48 hours. (a) NLRP3 mRNA; (b) ASC mRNA; (c) caspase-1 mRNA; (d) IL-18 mRNA; (e) IL-1β mRNA; (f) intracellular IL-18 protein levels; (g) intracellular IL-1β protein levels. Data were presented as the mean ± standard error (SE) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01, compared between the high-glucose and high-glucose+NLRP3 siRNA groups. There was no statistical significance between the glucose group and the NLRP3 siRNA group (low glucose: 5.5 mM, high glucose: 25 mM).

Figure 6

H₂S decreased high glucose-induced ROS production and inflammatory response in RPE cells. Intracellular ROS formation and inflammasome marker NLRP3, ASC, and caspase-1 expression were determined in ARPE-19 cells after 48 hours of culture under low and high glucose, with or without 200 μM of NaHS pretreatment. (a) The ROS production indicated by FACs in high-glucose culture; (b) the ROS production indicated by FACs in high-glucose+NaHS culture; (c) a bar graph of average results from the three individual experiments; (d) NLRP3 mRNA; (e) ASC mRNA; (f) caspase-1 mRNA. Data were presented as the mean ± standard error (SE) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01, compared between the high-glucose and high-glucose+H₂S groups (low glucose: 5.5 mM, high glucose: 25 mM).

Figure 7

H₂S decreased high glucose-induced proinflammatory cytokine IL-18 and IL-1β production in RPE cells. ARPE-19 cells were cultured in high glucose with or without 200 μM of NaHS pretreatment. The mRNA and intracellular protein expression levels were measured, accordingly. (a) IL-18 mRNA; (b) IL-1β mRNA; (c) intracellular IL-18 protein levels; (d) intracellular IL-1β protein levels. Data were presented as the mean ± standard error (SE) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01, compared between the low-glucose and high-glucose groups (low glucose: 5.5 mM, high glucose: 25 mM).